scholarly journals Functional analysis of the T-cell-restricted protein tyrosine kinase Txk

1998 ◽  
Vol 335 (2) ◽  
pp. 277-284 ◽  
Author(s):  
Jonathan H. ELLIS ◽  
Roger P. M. SUTMULLER ◽  
Martin J. SIMS ◽  
Susan COOKSLEY
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Lymphocyte Function ◽  
Signalling Molecules ◽  
Tec Kinases ◽  
A Tec

T lymphocytes express a range of tyrosine kinases that are involved in signalling processes driving cell activation, proliferation and differentation. Two tyrosine kinases expressed only in T cells, the Itk/Emt and Txk gene products, are members of the Tec family of kinases. The role of Tec kinases in cellular function is poorly understood, although a Tec kinase specific to B cells, Btk, is essential for B-cell development. To explore the contribution of the T-cell-specific Tec kinases to lymphocyte function, we have expressed human Txk in the baculovirus system and conducted the first characterization of its activity. We find that Txk exhibits a substrate preference in vitro quite distinct from that of the major T-cell kinases Lck and ZAP70, suggesting that Tec-family kinases might act on a distinct range of substrates. We also investigated the interactions of Txk with the cytoplasmic domains of the key signalling molecules CD3ζ, CD28 and CTLA4 and find that none of these are phosphorylated by Txk, nor are they ligands for the SH2 or SH3 domains of Txk. We conclude that it is unlikely that Txk has a role in the early signal transduction events associated with these key pathways controlling T-cell activation.

scholarly journals PAH- and PCB-induced Alterations of Protein Tyrosine Kinase and Cytokine Gene Transcription in Harbor Seal (Phoca Vitulina) PBMC

2005 ◽  
Vol 12 (2) ◽  
pp. 91-97 ◽  
Author(s):  
Jennifer C. C. Neale ◽  
Thomas P. Kenny ◽  
Ronald S. Tjeerdema ◽  
M. Eric Gershwin
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Cell Activation ◽  
Critical Role ◽  
Harbor Seal ◽  
Lymphocyte Function ◽  
Altered Expression ◽  
Protein Tyrosine

Mechanisms underlyingin vitroimmunomodulatory effects of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were investigated in harbor seal peripheral leukocytes, via real-time PCR. We examined the relative genetic expression of the protein tyrosine kinases (PTKs)FynandItk, which play a critical role in T cell activation, and IL-2, a cytokine of central importance in initiating adaptive immune responses. IL-1, the macrophage-derived pro-inflammatory cytokine of innate immunity, was also included as a measure of macrophage function. Harbor seal PBMC were exposed to the prototypic immunotoxic PAH benzo[a]pyrene (BaP), 3,3',4,4',5,5'-hexachlorobiphenyl (CB-169), a model immunotoxic PCB, or DMSO (vehicle control). Exposure of Con A-stimulated harbor seal PBMC to both BaP and CB-169 produced significantly altered expression in all four targets relative to vehicle controls. The PTKsFynandItkwere both up-regulated following exposure to BaP and CB-169. In contrast, transcripts for IL-2 and IL-1 were decreased relative to controls by both treatments. Our findings are consistent with those of previous researchers working with human and rodent systems and support a hypothesis of contaminant-altered lymphocyte function mediated (at least in part) by disruption of T cell receptor (TCR) signaling and cytokine production.

scholarly journals Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Matthias Kästle ◽  
Camilla Merten ◽  
Roland Hartig ◽  
Thilo Kaehne ◽  
Ardiyanto Liaunardy-Jopeace ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Enzymatic Activity ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Sh2 Domain ◽  
Jurkat T Cells

Abstract Background Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. Methods Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. Results Co-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation. Conclusions Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR. Major findings Our data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation.

scholarly journals Cd2 Sets Quantitative Thresholds in T Cell Activation

1999 ◽  
Vol 190 (10) ◽  
pp. 1383-1392 ◽  
Author(s):  
Martin F. Bachmann ◽  
Marijke Barner ◽  
Manfred Kopf
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Dose Response ◽  
Response Curve ◽  
Cell Activation ◽  
Dose Response Curve ◽  
Lymphocyte Function

It has been proposed that CD2, which is highly expressed on T cells, serves to enhance T cell–antigen presenting cell (APC) adhesion and costimulate T cell activation. Here we analyzed the role of CD2 using CD2-deficient mice crossed with transgenic mice expressing a T cell receptor specific for lymphocytic choriomeningitis virus (LCMV)-derived peptide p33. We found that absence of CD2 on T cells shifted the p33-specific dose–response curve in vitro by a factor of 3–10. In comparison, stimulation of T cells in the absence of lymphocyte function–associated antigen (LFA)-1–intercellular adhesion molecule (ICAM)-1 interaction shifted the dose–response curve by a factor of 10, whereas absence of both CD2–CD48 and LFA-1–ICAM-1 interactions shifted the response by a factor of ∼100. This indicates that CD2 and LFA-1 facilitate T cell activation additively. T cell activation at low antigen density was blocked at its very first steps, as T cell APC conjugate formation, TCR triggering, and Ca2+ fluxes were affected by the absence of CD2. In vivo, LCMV-specific, CD2-deficient T cells proliferated normally upon infection with live virus but responded in a reduced fashion upon cross-priming. Thus, CD2 sets quantitative thresholds and fine-tunes T cell activation both in vitro and in vivo.

scholarly journals Functional evidence for TCR-intrinsic specificity for MHCII

2016 ◽  
Vol 113 (11) ◽  
pp. 3000-3005 ◽  
Author(s):  
Heather L. Parrish ◽  
Neha R. Deshpande ◽  
Jelena Vasic ◽  
Michael S. Kuhns
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Specific Protein ◽  
Antigenic Peptides ◽  
Complex Molecules ◽  
Peptide Specificity ◽  
Contact Residues

How T cells become restricted to binding antigenic peptides within class I or class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) via clonotypic T-cell receptors (TCRs) remains debated. During development, if TCR–pMHC interactions exceed an affinity threshold, a signal is generated that positively selects the thymocyte to become a mature CD4+ or CD8+ T cell that can recognize foreign peptides within MHCII or MHCI, respectively. But whether TCRs possess an intrinsic, subthreshold specificity for MHC that facilitates sampling of the peptides within MHC during positive selection or T-cell activation is undefined. Here we asked if increasing the frequency of lymphocyte-specific protein tyrosine kinase (Lck)-associated CD4 molecules in T-cell hybridomas would allow for the detection of subthreshold TCR–MHC interactions. The reactivity of 10 distinct TCRs was assessed in response to selecting and nonselecting MHCII bearing cognate, null, or “shaved” peptides with alanine substitutions at known TCR contact residues: Three of the TCRs were selected on MHCII and have defined peptide specificity, two were selected on MHCI and have a known pMHC specificity, and five were generated in vitro without defined selecting or cognate pMHC. Our central finding is that IL-2 was made when each TCR interacted with selecting or nonselecting MHCII presenting shaved peptides. These responses were abrogated by anti-CD4 antibodies and mutagenesis of CD4. They were also inhibited by anti-MHC antibodies that block TCR–MHCII interactions. We interpret these data as functional evidence for TCR-intrinsic specificity for MHCII.

scholarly journals A Multivalent ICAM-1 Binding Nanoparticle which Inhibits ICAM-1 and LFA-1 Interaction Represents a New Tool for the Investigation of Autoimmune-Mediated Dry Eye

2020 ◽  
Vol 21 (8) ◽  
pp. 2758 ◽  
Author(s):  
Pang-Yu Hsueh ◽  
Yaping Ju ◽  
Adrianna Vega ◽  
Maria C. Edman ◽  
J. Andrew MacKay ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Dry Eye ◽  
Cell Activation ◽  
Intercellular Adhesion Molecule ◽  
In Vitro Assays ◽  
Lymphocyte Function ◽  
Loss Of Function ◽  
Mixed Cell

The autoimmune disorder, Sjögren’s syndrome (SS), is characterized by lymphocytic infiltration and loss of function of exocrine glands such as the lacrimal gland (LG) and salivary gland. SS-associated changes in the LG are associated with the development of autoimmune-mediated dry eye disease. We have previously reported the accumulation of intercellular adhesion molecule 1 (ICAM-1) in the LG of Non-Obese Diabetic (NOD) mice, a murine model of autoimmune-mediated dry eye in SS, in both LG acinar cells and infiltrating lymphocytes. ICAM-1 initiates T-cell activation and can trigger T-cell migration through binding to lymphocyte function-associated 1 antigen (LFA). To modulate this interaction, this study introduces a new tool, a multivalent biopolymeric nanoparticle assembled from a diblock elastin-like polypeptide (ELP) using the S48I48 (SI) ELP scaffold fused with a mouse ICAM-1 targeting peptide to form IBP-SI. IBP-SI forms a multivalent, monodisperse nanoparticle with a radius of 21.9 nm. Unlike the parent SI, IBP-SI binds mouse ICAM-1 and is internalized by endocytosis into transfected HeLa cells before it accumulates in lysosomes. In vitro assays measuring lymphocyte adhesion to Tumor Necrosis Factor TNF-α-treated bEnd.3 cells, which express high levels of ICAM-1, show that adhesion is inhibited by IBP-SI but not by SI, with IC50 values of 62.7 μM and 81.2 μM, respectively, in two different assay formats. IBP-SI, but not SI, also blocked T-cell proliferation in a mixed lymphocyte reaction by 74% relative to proliferation in an untreated mixed cell reaction. These data suggest that a biopolymeric nanoparticle with affinity for ICAM-1 can disrupt ICAM-1 and LFA interactions in vitro and may have further utility as an in vivo tool or potential therapeutic.

scholarly journals A negative role for the interleukin-2-inducible T-cell kinase (ITK) in human Foxp3+ TREG differentiation

2018 ◽  
Author(s):  
Polina Mamontov ◽  
Ryan A. Eberwine ◽  
Jackie Perrigoue ◽  
Anuk Das ◽  
Joshua R. Friedman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Kinase Inhibitor ◽  
Interleukin 2 ◽  
Tec Kinases ◽  
Human T Cells ◽  
Inducible T Cell Kinase

ABSTRACTThe Tec kinases ITK (interleukin-2-inducible T-cell kinase) and RLK (resting lymphocyte kinase) are critical components of the proximal TCR/CD3 signal transduction machinery, and data in mice suggest that ITK negatively regulates TREG differentiation. However, whether Tec kinases modulate TREG development and/or function in human T cells remains unknown. Using a novel self-delivery siRNA platform (sdRNA), we found that ITK knockdown in primary human naïve peripheral blood CD4 T cells increased Foxp3+ TREG differentiation under both TREG and T effector (Teff) cell priming conditions. ITK knockdown also enhanced the expression of the co-inhibitory receptor PD-1 on FoxP3+ T cells. TREGS differentiated in vitro (iTREG) after ITK knockdown displayed suppressive capacity against effector CD4+ T cell proliferation. ITK knockdown decreased IL-17A production in T cells primed under Th17 conditions and increased Th1 differentiation. Finally, a dual ITK/RLK Tec kinase inhibitor blocked TREG differentiation and T cell activation in general. Our data suggest that targeting ITK in human T cells may be an effective approach to boost TREG in the context of autoimmune diseases, but non-specific inhibition of other Tec family kinases may broadly inhibit T cell activation.

scholarly journals T Cell-Derived IL-17A Induces Vascular Dysfunction via Perivascular Fibrosis Formation and Dysregulation of ⋅NO/cGMP Signaling

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Rebecca Schüler ◽  
Panagiotis Efentakis ◽  
Johannes Wild ◽  
Jérémy Lagrange ◽  
Venkata Garlapati ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Vascular Function ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Vascular Dysfunction ◽  
Myeloid Cells ◽  
Tyrosine Kinase 2 ◽  
The Impact

Aims. The neutrophil recruiting cytokine Interleukin-17A (IL-17A) is a key component in vascular dysfunction and arterial hypertension. Moreover, IL-17A has a central role for the vascular infiltration of myeloid cells into the arterial wall in Angiotensin II-induced vascular inflammation. The intention of our study was to analyze the impact of T cell-derived IL-17A on hypertension, vascular function, and inflammation. Methods and Results. Chronic IL-17A overexpression in T cells (CD4-IL-17Aind/+ mice) resulted in elevated reactive oxygen species in the peripheral blood and a significant vascular dysfunction compared to control mice. The vascular dysfunction seen in the CD4-IL-17Aind/+ mice was only accompanied by a modest and nonsignificant accumulation of inflammatory cells within the vessel wall. Therefore, infiltrating myeloid cells did not serve as an explanation of the vascular dysfunction seen in a chronic IL-17A-driven mouse model. In addition to vascular dysfunction, CD4-IL-17Aind/+ mice displayed vascular fibrosis with highly proliferative fibroblasts. This fibroblast proliferation was induced by exposure to IL-17A as confirmed by in vitro experiments with primary murine fibroblastic cells. We also found that the ⋅NO/cGMP pathway was downregulated in the vasculature of the CD4-IL-17Aind/+ mice, while levels of protein tyrosine kinase 2 (PYK2), an oxidative stress-triggered process associated with T cell activation, were upregulated in the perivascular fat tissue (PVAT). Conclusions. Our data demonstrate that T cell-derived IL-17A elicits vascular dysfunction by mediating proliferation of fibroblasts and subsequent vascular fibrosis associated with PYK2 upregulation.

scholarly journals CRL4-DCAF12 Ubiquitin Ligase Controls MOV10 RNA Helicase during Spermatogenesis and T Cell Activation

2021 ◽  
Vol 22 (10) ◽  
pp. 5394
Author(s):  
Tomas Lidak ◽  
Nikol Baloghova ◽  
Vladimir Korinek ◽  
Radislav Sedlacek ◽  
Jana Balounova ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Ubiquitin Ligase ◽  
Cell Activation ◽  
Rna Helicase ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Risc Complex ◽  
Wide Range

Multisubunit cullin-RING ubiquitin ligase 4 (CRL4)-DCAF12 recognizes the C-terminal degron containing acidic amino acid residues. However, its physiological roles and substrates are largely unknown. Purification of CRL4-DCAF12 complexes revealed a wide range of potential substrates, including MOV10, an “ancient” RNA-induced silencing complex (RISC) complex RNA helicase. We show that DCAF12 controls the MOV10 protein level via its C-terminal motif in a proteasome- and CRL-dependent manner. Next, we generated Dcaf12 knockout mice and demonstrated that the DCAF12-mediated degradation of MOV10 is conserved in mice and humans. Detailed analysis of Dcaf12-deficient mice revealed that their testes produce fewer mature sperms, phenotype accompanied by elevated MOV10 and imbalance in meiotic markers SCP3 and γ-H2AX. Additionally, the percentages of splenic CD4+ T and natural killer T (NKT) cell populations were significantly altered. In vitro, activated Dcaf12-deficient T cells displayed inappropriately stabilized MOV10 and increased levels of activated caspases. In summary, we identified MOV10 as a novel substrate of CRL4-DCAF12 and demonstrated the biological relevance of the DCAF12-MOV10 pathway in spermatogenesis and T cell activation.

scholarly journals CD14 Expressing Precursors Give Rise to Highly Functional Conventional Dendritic Cells for Use as Dendritic Cell Vaccine

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3818
Author(s):  
Maud Plantinga ◽  
Denise A. M. H. van den Beemt ◽  
Ester Dünnebach ◽  
Stefan Nierkens
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Cord Blood ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Dendritic Cell Vaccine ◽  
Cell Vaccine ◽  
Activated T Cells

Induction of long-lasting immunity by dendritic cells (DCs) makes them attractive candidates for anti-tumor vaccination. Although DC vaccinations are generally considered safe, clinical responses remain inconsistent in clinical trials. This initiated studies to identify subsets of DCs with superior capabilities to induce effective and memory anti-tumor responses. The use of primary DCs has been suggested to overcome the functional limitations of ex vivo monocyte-derived DCs (moDC). The ontogeny of primary DCs has recently been revised by the introduction of DC3, which phenotypically resembles conventional (c)DC2 as well as moDC. Previously, we developed a protocol to generate cDC2s from cord blood (CB)-derived stem cells via a CD115-expressing precursor. Here, we performed index sorting and single-cell RNA-sequencing to define the heterogeneity of in vitro developed DC precursors and identified CD14+CD115+ expressing cells that develop into CD1c++DCs and the remainder cells brought about CD123+DCs, as well as assessed their potency. The maturation status and T-cell activation potential were assessed using flow cytometry. CD123+DCs were specifically prone to take up antigens but only modestly activated T-cells. In contrast, CD1c++ are highly mature and specialized in both naïve as well as antigen-experienced T-cell activation. These findings show in vitro functional diversity between cord blood stem cell-derived CD123+DC and CD1c++DCs and may advance the efficiency of DC-based vaccines.

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